2. Lecture Epithelia and Glands

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Epithelia and Glands

Jean-Pierre Louboutin

7/9/12

Jean-Pierre Louboutin

- Neurologist

- MD: dissertation on correlations between clinical, neurophysiological and neuroradiological data in Multiple Sclerosis

- PhD: dissertation on morphological and physiological properties during skeletal muscle regeneration

- Research in USA (Philadelphia) focused on gene/cell therapy/immunology (University of Pennsylvania, UPENN, 6 years) and also on physiopathology and molecular therapy of HIV-1-associated dementia (Thomas Jefferson University, 7 years)

CellBasic structural and functional unit of all living organism but

Tissue..Humans are not unicellular organisms and cells are organized in humans as tissues, ensemble of similar cells of the same origin, that together carry out a specific function- Study of tissues: histology+ Epithelia

- Definitions- Functions- Different types- Special cytological features

+ Glands

- Definitions and functions- Different types

+ Different staining methods in histology

+ Examples of experimental therapies targeting epithelia

- Gene transfer- Regenerative medicine

+ Objectives+ Few words of advice+ Epithelia


+ Glands





+ Objectives+ Few words of advice

Four basic types of animal tissues:

. Epithelia

. Connective tissue

. Muscle tissue

. Nervous tissueDefinitions. Epithelia: tissues consisting of closely apposed cells, without intervening intercellular substances

. Epithelia are avascular but innervated; all epithelia "grow" on an underlying layer of vascular connective tissue

. The connective tissue and the epithelium are separated by abasement membrane

. Epithelium covers all free surfaces of the body (i.e., skin) . Epithelium also lines the large internal body cavities, where it is termedmesothelium

. The internal surfaces of blood and lymph vessels are lined by epithelium, here calledendotheliumFunctions of epithelia. Protection from toxins, physical trauma, radiation (e.g., skin)

. Absorption: cells of the gastrointestinal tract can absorb nutrients from the digestive tract lumen (lumen: inside space of a tubular structure)

. Excretion: epithelial tissue lines the excretory lumina like the urinary tracts from the kidneys through the urethra

. Secretion of sweat, mucus, enzymes delivered by ducts (channels/canals) of the glandular epithelium

. Epithelia are classified on the basis of the number of cell layers and the shape of the cellsin the surface layer

. If there is only one layer of cells in the epithelium, it is designatedsimple

. If there are two or more layers of cells, it is calledstratified

. Cells in the surface layer are described according to their height assquamous(scale- or plate-like),cuboidalorcolumnar

Types of epithelia

Simple squamous epithelium. Cells: single layer, flattened. Nuclei: flattened, centrally located. Functions: diffusion, lubrication. Examples: blood vessels, heart lined by simple squam. epithelium

Simple cuboidal epithelium. Cells: single square-like layer. Nuclei: centrally located and spherical. Functions: absorption, secretion, protection. Examples: tubules of kidney, follicles of thyroid, surfaces of ovaries, excretory ducts of many glands

Simple columnar epithelium. Cells: tall, single-layered. Nuclei: basally located and elongated. Functions: absorption, protection, secretion (goblet cells with microvilli, cilia). Examples: gastrointestinal tract (cardia of stomach to rectum)

Stratified squamous epithelium (1). Cells: squamous cells apically (at the top), but basal layers vary from cuboidal to columnar. Nuclei: centrally located. Function: protection. Examples: skin, oesophagus

Stratified squamous keratinized epithelium: skinStratified squamous epithelium (2). Cells: squamous cells apically (at the top), but basal layers vary from cuboidal to columnar. Nuclei: centrally located. Function: protection. Examples: skin, oesophagus

Stratified squamous non-keratinized epithelium: oesophagus

Stratified cuboidal epithelium. Cells: two layers. Nuclei: centrally located and spherical. Functions: absorption, secretion. Examples: duct of the sweat glands

Stratified columnar epithelium. Cells: single layer of columnar cells on one or several layers of cuboidal cells. Nuclei: basal and oval. Functions: protection, secretion. Examples: duct of the mammary gland and large salivary glands

Transitional epithelium. Cells: apical cells often large, round and bi-nucleated. Nuclei: centrally located. Functions: distention. Examples: bladder, ureter and urethra

Pseudostratified columnar epithelium. Cells: differ in height, not all cells reach apical surface. Nuclei: at various positions. Functions: absorption, secretion, transportation (goblet cells, cilia). Examples: trachea, excretory ducts (canals) of many glands

TracheaPseudostratified columnar epitheliumTerminal bronchioleSimple columnar epitheliumBronchusPseudostratified columnarepitheliumAlveoliSquamous epithelial cells (type I pneumocytes)Different epithelia can be seen in the same organ

Respiratory bronchioleSimple cuboidal epitheliumSpecial cytological features of epitheliaBasement membrane (BM):

. Epithelia separated from underlying connective tissue by extracellular layer: BM

. BM acts as a scaffolding on which epithelium can grow and regenerate after injury

. BM composed of 2 sublayers:

1) Basal lamina (80 nm thick) consists of protein filaments embedded in amorphous matrix. Major components of basal lamina are two glycoproteins: laminin and collagenBasal lamina: selectively permeable filter between epithelium and connective tissue

2) Reticular lamina consists of reticular fibres embedded in ground substance. Fibres of the reticular lamina connect basal lamina with underlying connective tissue. Contains fibronectin

EpitheliumBasement MembraneConnective TissueBasal LaminaReticular LaminaConnective TissueSpecial features of the apical surface:

. Microvilli and stereocilia are finger-shaped extensions of epithelial cells. Function: to increase the surface area of epithelial cells. Found in epithelia active in absorption. Microvilli contain actin filaments

. Microvilli are much shorter than stereocilia. Stereocilia are not moving structures

. Goblet cells: mucus-secreting cells found in pseudo-stratified and columnar epithelia (e.g., gastro-intestinal tract and trachea)

. Cilia can help move molecules within the lumen. Found in pseudostratified and columnar epithelia (e.g., trachea)

Miicrovilli

*

Isolated cellTissueNature of the junction between 2 cells ?Special features of the basal and lateral surfaces: cell junctions

. Multicellular organisms require adhesion for cells to adhere to each other or to the extracellular matrix

. Cell adhesion occurs through specific cellular specializations and molecules and has both static and dynamic functions

. Cell junctions are especially abundant in epithelia

. 3 major types of cell junctions:+ Gap junctions: communicating junctions+ Adherens junctions, desmosome, hemidesmosome: anchoring junctions+ Tight junctions: occluding junctions

. Gap junctions connect cytoplasm of 2 cells and are made up of proteins called connexins

. Desmosomes: Group of glycoproteins (cadherins) inserted into the opposing plasma membrane (like a zipper linking both cells)- Mediate cell-to-cell adhesion

. Hemidesmosomes resemble desmosomes but are made up of integrins (transmembrane protein) instead of cadherin

Gap junctionsDesmosomesCommunicating junctionsAnchoring junctions. Zonula adherens (adherens junctions): cytoplasmic face is linked to the actin cytoskeleton. They can appear as bands encircling the cell or as spots of attachment to the extracellular matrix (alpha and beta catenins links to actin filaments and transmembrane anchors are cadherins)

. Tight junctions (zonula occludens): isolate the interior of the body from the outside world. Proteins inserted into the cell membranes of adjacent cells 'stitch' the membranes of the cells together. Provide an effective barrier to the diffusion of substances from the outside of the epithelium (i.e., occludin, claudin-1)

Tight junctionOccluding junctions+ Epithelia


+ Glands






. Glands are aggregations of cells whose function is secretion

. Exocrine glandsrelease the secretory product via ducts (channels) that open upon one of the surfaces of the body in contact with the external world (gastrointestinal tract, etc.). Endocrine glandsrelease their secretory product (typically hormones) into the spaces between the secretory cells (extracellular space) from which it enters the bloodstream

. Both endocrine and exocrine glands are developmentally derived from epithelia. Exocrine glands maintain the connection with the surface epithelium, whereas the connection is lost by endocrine glandsGlands: definitions and functions. Exocrine glands may be classified according to cell number, and/or the shape and branching pattern of their secretory portions and ducts

. Unicellular or multicellular describes a single-celled gland or a gland made of many cells, respectively . A multicellular gland consists of a group of secretory cells and a duct through which the secretions pass as they exit the gland

. Branched glands refers to the branching arrangement of secretory cells

. Simple or compound refers to whether the duct of the gland (not the secretory cells) does or does not branch, respectively

. Tubular describes a gland whose secretory cells form a tube, while alveolar (or acinar) describes secretory cells that form a bulblike sac. Combinations exist : the pancreas is a tubulo-acinar gland

Different types of exocrine glandsUnicellular glands. Consist of a single secretory cell

. In mammals, the only example of unicellular exocrine glands are goblet cells, which are found in the epithelium of many mucous membranes

. Goblet cells secrete the glycoprotein mucin, which by the uptake of water, is converted into a slimy substance, mucus

JejunumRectum

. The simplest form of a multicellular gland is asecretory epithelial sheath

. It is a surface epithelium consisting entirely of secretory cells(e.g., the epithelium lining the inner surface of the stomach, where the mucous secretion protects the stomach wall from the acidic contents of the stomach)Multicellular glandsAcidic content. Other multicellular glands have their secretory portion embedded in the connective tissue underlying the epithelium

. The secretion is either discharged directly from the secretory portion onto the epithelium or reaches the epithelium via a duct system that consists of non-secretory cells

. The secretory portion may have a variety of shapes. Secretory cells may form:+ tubesintubular glands,+ aciniinacinar glandsor+ alveoliinalveolarglands

. Branched glands refers to the branching arrangement of cells

. Simple or compound refers to whether the duct of the gland (not the secretory portion) does or does not branch, respectively

Exocrine glands are also classified according to their function:. In merocrine glands, secretions pass through the cell membranes of the secretory cells (exocytosis). For example, goblet cells of the trachea release mucus via exocytosis

. In apocrine glands, a portion of the cell containing secretions is released as it separates from the rest of the cell. For example, the apical portion of lactiferous glands release milk in this manner

. In holocrine glands, entire secretory cells disintegrate and are released along with their contents. For example, sebaceous glands release sebum to lubricate the skin

. Two morphologically distinct forms:serousormucous . Serous secretions have a low viscosity, i.e. they are rather "watery". The apical cytoplasm of the cells formingserous aciniis usually well-stained

. Mucous secretions have a high viscosity, i.e. they are rather "slimy".The contents of the secretory vesicles in the apical cytoplasm of cells ofmucous aciniare only weakly stained, giving a foamy appearance Serous and mucous acini

. Endocrine glandssecretehormonesdirectly into the bloodstream.

. For example, the thyroid gland secretes the hormone thyroxin into the bloodstream, where it is distributed throughout the body, stimulating an increase in the metabolic rate of body cells.Endocrine glands

+ Epithelia


+ Glands






. Hematoxylin and eosin (H&E) staining

. H&E stains nuclei in dark blue and cytoplasm and other structures in pink/red

. Example: trachea

. Other methods can be used to see molecular and structuralcomponents of the trachea

Different staining methods

. Immunohistochemistry: technique using antibodies to target specific proteins or peptides antigens in the cell

. Bound antibodies can be detected using several methods (e.g., fluorophores)

. Example: detection of a molecule involved in innate immune system, Toll-like receptor 2 (TLR2) in the apex of the trachea

TLR2TLR2TLR2TLR2ControlControlLow Magn.High Magn.

TLR5Mucin-1TLR5 + Mucin-1 + DAPI (nuclei)TLR5TLR5Control. Double Immunohistochemistry: technique allowing to detect 2 different antigens in a cell/tissue. Ex: detection of another molecule, Toll-like receptor 5 (TLR5) and mucin-1 (present in goblet cells) in the trachea

. Histochemistry: identification and localization of molecular components, metabolic and enzymatic activities in tissues. Example: gene transfer of the lac Z gene into the trachea usinga viral vector. Lac Z gene encodes for beta-galactosidase enzyme. Staining for beta-galactosidase enzymatic activity: proof of gene transfer

Lac Z geneVirusControlBeta-GalactosidaseBeta-Galactosidase

Electron Microscopy (EM): provides high-powered resolution imagesTransmission EM (TEM)Scanning EM (TEM)+ Epithelia


+ Glands






Examples of experimental therapies targeting epitheliaGene therapy based on the use of DNA as a pharmaceutical agent to treat a disease

Most common form of gene therapy involves using DNA that encodes a therapeutic gene in order to replace amutated gene (i.e., sickle cell anemia, cystic fibrosis) Other forms involve using DNA that encodes a therapeutic protein drug (rather than a natural human gene) to provide treatment

In gene therapy, DNA that encodes a therapeutic protein is packaged within a "vector", usually a virus, which is used to get the DNA inside cells of the body. Once inside the cell, the DNA becomes expressed by the cell machinery, resulting in the production of therapeutic protein, which in turn treats the patient's diseaseGene transfer/Gene therapyVirusAU1 transgene

DAPI: staining of nucleiStaining of transgene AU1 in alveolar cellsMerged DAPI + AU1Alveoli. Gene delivery to tissues/organs

. Virus can target tissues, leading to gene delivery and transgene expressionRegenerative medicineRegenerative medicineis the process of replacing or regenerating human cells, tissues or organs to restore or establish normal function

This field holds the promise of regenerating damaged tissues and organs in the body by replacing damaged tissue and/or by stimulating the body's own repair mechanisms to heal previously irreparable tissues or organs

Potential to solve the problem of the shortage of organs available for donation

Refers to a group of biomedical approaches to clinical therapies thatmay involve the use ofstem cells

Examples include the injection ofstem cells or progenitor cells (cell therapies); the induction ofregeneration by biologically active molecules administered alone or as a secretion by infused cells (immunomodulation therapy); andtransplantation ofin vitrogrown organs and tissues (tissue engineering)

TransgeneVirusBM stem cellsStudy of the distribution of the transgene in the bodyMigration of BM stem cells ?Targeting of bone marrow (BM) stem cells in vivo by direct injectioninto the femoral cavity

Using a viral vector carrying a transgeneFollowing the distribution of BM stem cells in the body by studying transgene expression

Louboutin et al., J Gene Med, 2007Transgene expression in the rat lung at the alveolar level

- DAPI staining of nuclei; FLAG: transgene; - TTF-1: marker of precursor cells in the lungsTransgene expression in the rabbit lung at the alveolar level

- DAPI staining of nuclei; AU1: transgene; - TTF-1: marker of precursor cells in the lungs

Conclusions

Bone marrow stem cells can be transduced in situ in the BM

They migrate to the lungs

They differentiate into precursor cells in the lungs

Study of capillaries of the rat brain after BM injection of a viral vector carrying the gene for the transgene FLAG

DAPI: marker of nuclei; FLAG: transgene; CD31: marker of endothelial cells and capillaries

Study of capillaries of the rabbit brain after BM injection of a viral vector carrying the gene for the transgene AU1

- DAPI: marker of nuclei; AU1: transgene; CD31: marker of endothelial cells and capillariesConclusionsBM stem cells can migrate to the brain vessels

They can differentiate into endothelial cellsLouboutin et al., Neuroscience, 2011+ Epithelia


+ Glands





+ Objectives+ Few words of adviceObjectives- To know the major features of epithelial tissue ++

To know the different types of epithelial tissues (including the corresponding organs) +++

- To know the special cytological features of epithelia ++

To know the different types of glandular tissues (including the corresponding organs) ++++ Epithelia


+ Glands





+ Objectives+ Few words of adviceFew words of advice- Attend the lectures: + It helps for memorizing the topics and for putting things in order+ Take notes+ Better for getting hints about the exams

- Stick to the lectures rather than to the books:

+ Do not get overwhelmed by the books+ Use the books as references to check the lectures or the points not understood during the lectures+ Stick to the essential at first; enrich your knowledge with details progressively

- Read and reread the lectures while taking notes or making drawings:

+ Make sure to work on the lectures regularly and to memorize thelectures at the end of each week+ 4 hours each day in average til mid November (for all fields)+ 6-8 hours a day in average from mid November til the exams

- Spend time in the Laboratory bringing lectures and/or books

- Do not be afraid to ask questions or ask for help +++

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2. Lecture Epithelia and Glands